Method of production of L-amino acids

ABSTRACT

An isolated polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, with the L-aspartic acid at position 5 of the amino acid sequence replaced by another proteinogenic amino acid, and possesses citrate synthase activity. In addition, a vector comprises the polynucleotide and a bacterium comprises the vector. An isolated polynucleotide comprises a nucleotide sequence comprising, from position 1 to 39, the nucleotide sequence corresponding to position 1 to 39 of SEQ ID NO: 11, from position 40 to 105, a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 12, with each proteinogenic amino acid except L-aspartic acid being present at position 5. A method of producing an L-amino acids is also described.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to novel polynucleotides coding for a polypeptidewith citrate synthase activity, bacteria containing the polynucleotidesand polypeptides and methods of production of amino acids using thesebacteria.

2. Discussion of the Background

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.Further, the materials, methods, and examples are illustrative only andare not intended to be limiting, unless otherwise specified.

Amino acids are used in human medicine, in the pharmaceutical industry,in the food industry and quite particularly in animal nutrition.

Amino acids may be produced by fermentation of strains of coryneformbacteria, preferably Corynebacterium glutamicum. Owing to their greatimportance, work is constantly in progress for improving the productionprocesses. Process improvements may relate to the fermentationtechnology, for example, stirring and supply of oxygen, or to thecomposition of the nutrient media, for example, the sugar concentrationduring fermentation, or processing to the product form by, for example,ion-exchange chromatography, or to the intrinsic performancecharacteristics of the microorganism itself.

Methods of mutagenesis, selection and mutant screening are employed forimproving the performance characteristics of these microorganisms. Inthis way we obtain strains that are resistant to antimetabolites or areauxotrophic for metabolites of regulatory importance, and which produceamino acids. A known antimetabolite is the lysine analogS-(2-aminoethyl)-L-cysteine (AEC).

Methods of recombinant DNA technology have also been used for some yearsnow for strain improvement of L-amino acid-producing strains of thegenus Corynebacterium, preferably Corynebacterium glutamicum, byamplifying individual amino acid biosynthesis genes and investigatingthe effect on amino acid production.

Synoptic descriptions of the biology, genetics and biotechnology ofCorynebacterium glutamicum are given in “Handbook of Corynebacteriumglutamicum” (Eds.: L. Eggeling and M. Bott, CRC Press, Taylor & Francis,2005), in the special issue of the Journal of Biotechnology (ChiefEditor: A. Pühler) with the title “A new era in Corynebacteriumglutamicum biotechnology” (Journal of Biotechnology 104/1-3, (2003)) andin the book by T. Scheper (Managing Editor) “Microbial Production ofL-Amino Acids” (Advances in Biochemical Engineering/Biotechnology 79,Springer Verlag, Berlin, Germany, 2003).

The nucleotide sequence of the genome of Corynebacterium glutamicum isdescribed in Ikeda and Nakagawa (Applied Microbiology and Biotechnology62, 99-109 (2003)), in EP 1 108 790 and in Kalinowski et al. (Journal ofBiotechnology 104/1-3, 2003)).

The nucleotide sequence of the genome of Corynebacterium efficiens isdescribed in Nishio et al. (Genome Research, 13 (7), 1572-1579 (2003)).

The nucleotide sequences of the genome of Corynebacterium glutamicum andCorynebacterium efficiens are also available in the database of theNational Center for Biotechnology Information (NCBI) of the NationalLibrary of Medicine (Bethesda, Md., USA), in the DNA Data Bank of Japan(DDBJ, Mishima, Japan) or in the nucleotide sequence database of theEuropean Molecular Biology Laboratories (EMBL, Heidelberg, Germany andCambridge, UK).

The wild-type sequence of the coding region of the gltA gene ofCorynebacterium glutamicum is presented in SEQ ID NO: 1 in thespecification of the present application. In addition, the sequenceslocated upstream and downstream of the coding region are shown in SEQ IDNO: 3 and 25. The amino acid sequence of the encoded GltA polypeptide(citrate synthase) is accordingly given in SEQ ID NOs: 2, 4 and 26.

SUMMARY OF THE INVENTION

It was an object of the present invention to provide novel measures forthe improved production of L-amino acids, preferably L-lysine, L-valineand L-isoleucine, and more preferably L-lysine.

This and other objects have been achieved by the present invention thefirst embodiment of which includes an isolated polynucleotide, encodinga polypeptide comprising the amino acid sequence of SEQ ID NO: 2,wherein the L-aspartic acid at position 5 of the amino acid sequence isreplaced by another proteinogenic amino acid and wherein the polypeptidepossesses citrate synthase activity.

The invention further provides a vector comprising the isolatedpolynucleotide and a bacterium that has been transformed with thevector.

The invention also provides a method of production of an L-amino acid.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE represents a map of the plasmid pK18mobsacB_gltAD5V.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an isolated polynucleotide that codes for apolypeptide which comprises the amino acid sequence of SEQ ID NO: 2,wherein the L-aspartic acid at position 5 of the amino acid sequence isreplaced by another proteinogenic amino acid, preferably L-valine,L-leucine and L-isoleucine, and more preferably L-valine, and whereinthe polypeptide possesses citrate synthase activity (EC No. 4.1.3.7).Optionally, the polypeptide comprises at least one conservative aminoacid substitution, with the citrate synthase activity of the polypeptidebeing essentially unchanged by the conservative amino acidsubstitutions.

Proteinogenic amino acids are understood as meaning the amino acids thatoccur in natural proteins, i.e. in proteins of microorganisms, plants,animals and humans. These include in particular L-amino acids, selectedfrom the group L-aspartic acid, L-asparagine, L-threonine, L-serine,L-glutamic acid, L-glutamine, glycine, L-alanine, L-cysteine, L-valine,L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine,L-histidine, L-lysine, L-tryptophan, L-proline and L-arginine.

The terms polypeptide and protein are used as synonyms.

The invention further relates to vectors and bacteria, preferably of thegenus Corynebacterium and Escherichia, and more preferably of thespecies Corynebacterium glutamicum and Escherichia coli, which containthe stated polynucleotide or were produced using the statedpolynucleotide.

The invention also relates finally to bacteria preferably of the genusCorynebacterium and Escherichia, and more preferably of the speciesCorynebacterium glutamicum and Escherichia coli, which have beentransformed with the stated vector.

The term transformation comprises all methods for transferringpolynucleotides, preferably DNA, into a desired bacterium. Among otherthings these include the use of isolated DNA in transformation,electrotransformation or electroporation, transfer by cellular contactas in conjugation or the transfer of DNA by particle bombardment.

A further aspect of the invention relates to a bacterium, that may be arecombinant bacterium, of the genus Corynebacterium, which comprises apolynucleotide that codes for a polypeptide with citrate synthaseactivity, which comprises the amino acid sequence of SEQ ID NO: 2,wherein each proteinogenic amino acid except L-aspartic acid, preferablyL-valine, L-leucine and L-isoleucine, and preferably L-valine, iscontained at position 5 of the amino acid sequence. Optionally, thepolypeptide may contain one or more conservative amino acidsubstitution(s), with the citrate synthase activity of the polypeptidebeing essentially unchanged by the conservative amino acidsubstitutions.

A further aspect of the invention relates to a method of production ofL-amino acids, preferably L-lysine, L-valine and L-isoleucine, and morepreferably L-lysine, comprising the following steps:

a) fermentation of the recombinant bacteria of the genus Corynebacteriumaccording to the invention in a suitable nutrient medium, and

b) accumulation of the L-amino acid in the nutrient medium or in thecells of the bacteria.

“L-amino acids” means the proteinogenic amino acids.

If L-lysine or lysine is mentioned hereinafter, this is intended to meannot only the bases, but also the salts, for example L-lysinemonohydrochloride or L-lysine sulfate.

With regard to the bacteria of the genus Corynebacterium, L-aminoacid-excreting strains are preferred, based on the following species:

Corynebacterium efficiens, for example the strain DSM44549,

Corynebacterium glutamicum, for example the strain ATCC13032,

Corynebacterium thermoaminogenes, for example the strain FERM BP-1539,and

Corynebacterium ammoniagenes, for example the strain ATCC6871, thespecies Corynebacterium glutamicum being preferred.

Some representatives of the species Corynebacterium glutamicum are alsoknown under different designations. Examples include:

Corynebacterium acetoacidophilum ATCC 13870,

Corynebacterium lilium DSM20137,

Corynebacterium melassecola ATCC 17965,

Brevibacterium flavum ATCC14067,

Brevibacterium lactofermentum ATCC 13869, and

Brevibacterium divaricatum ATCC14020.

Known representatives of amino acid-excreting strains of the genusCorynebacterium are, for example, the L-lysine producing strains:

Corynebacterium glutamicum DM58-1/pDM6 (=DSM4697) described in EP 0 358940,

Corynebacterium glutamicum MH20-22B (=DSM16835) described in Menkel etal. (Applied and Environmental Microbiology 55(3), 684-688 (1989)),

Corynebacterium glutamicum AHP-3 (=FERM BP-7382) described in EP 1 108790,

Corynebacterium glutamicum DSM16834 described in (PCT/EP2005/012417),

Corynebacterium glutamicum DSM17119 described in (PCT/EP2006/060851),

Corynebacterium glutamicum DSM17223 described in (PCT/EP2006/062010),

Corynebacterium glutamicum DSM16937 described in (PCT/EP2005/057216),and

Corynebacterium thermoaminogenes AJ12521 (=FERM BP-3304) described inU.S. Pat. No. 5,250,423;

or the L-valine producing strains:

-   -   Brevibacterium lactofermentum FERM BP-1763 described in U.S.        Pat. No. 5,188,948,    -   Brevibacterium lactofermentum FERM BP-3007 described in U.S.        Pat. No. 5,521,074,    -   Corynebacterium glutamicum FERM BP-3006 described in U.S. Pat.        No. 5,521,074, and    -   Corynebacterium glutamicum FERM BP-1764 described in U.S. Pat.        No. 5,188,948;        or the L-isoleucine producing strains:    -   Brevibacterium flavum FERM-BP 759 described in U.S. Pat. No.        4,656,135,    -   Corynebacterium glutamicum FERM-BP 757 described in U.S. Pat.        No. 4,656,135,    -   Brevibacterium flavum FERM-BP 760 described in U.S. Pat. No.        4,656,135,    -   Corynebacterium glutamicum FERM-BP 758 described in U.S. Pat.        No. 4,656,135,    -   Brevibacterium flavum FERM BP-2215 described in U.S. Pat. No.        5,705,370, and    -   Brevibacterium flavum FERM BP-2433 described in U.S. Pat. No.        5,705,370.

Information on the taxonomic classification of strains of this group ofbacteria can be found inter alia in Seiler (Journal of GeneralMicrobiology 129, 1433-1477 (1983)), Kämpfer and Kroppenstedt (CanadianJournal of Microbiology 42, 989-1005 (1996)), Liebl et al.(International Journal of Systematic Bacteriology 41, 255-260 (1991))and in U.S. Pat. No. 5,250,434.

Strains with the designation “ATCC” can be obtained from the AmericanType Culture Collection (Manassas, Va., USA). Strains with thedesignation “DSM” can be obtained from the Deutsche Sammlung vonMikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). Strainswith the designation “FERM” can be obtained from the National Instituteof Advanced Industrial Science and Technology (AIST Tsukuba Central 6,1-1-1 Higashi, Tsukuba Ibaraki, Japan). The strain Corynebacteriumthermoaminogenes (FERM BP-1539) is described in U.S. Pat. No. 5,250,434.

For production of the polynucleotides, it is possible to use classicalin-vivo mutagenesis techniques with cell populations of bacteria of thegenus Corynebacterium using mutagenic substances such asN-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or using ultraviolet light.Mutagenesis techniques are described for example in the Manual ofMethods for General Bacteriology (Gerhard et al. (Eds.), AmericanSociety for Microbiology, Washington, D.C., USA, 1981) or in Tosaka etal. (Agricultural and Biological Chemistry 42(4), 745-752 (1978)) or inKonicek et al. (Folia Microbiologica 33, 337-343 (1988)).

From the mutagenized cell population, those mutants are taken andmultiplied which require L-glutamic acid or citric acid in order to beable to grow on a minimal agar or whose growth on the minimal agar isimproved by adding L-glutamic acid or citric acid. It is also possible,starting from mutants requiring L-glutamic acid or citric acid, toisolate so-called revertants, which do not require L-glutamic acid orcitric acid for their growth. These L-glutamic acid-auxotrophic orcitric acid-auxotrophic mutants or their respective revertants are theninvestigated. Technical details on the isolation of mutants withdefective citrate synthase activity can be found for example in Shiio etal. (Agricultural and Biological Chemistry 46(1), 101-107 (1982)).

Next, DNA is prepared or isolated from the mutants and by means of, forexample, the polymerase chain reaction (PCR) using primer pairs whichallow the amplification of the gltA gene or gltA allele, thecorresponding polynucleotide is synthesized and isolated.

For this, it is possible to select any primer pairs from the nucleotidesequence located upstream and downstream of the coding region and thenucleotide sequence that is complementary to it (see SEQ ID NOs: 3 and25). A primer of a primer pair then comprises preferably at least 15, atleast 18, at least 20, at least 21 or at least 24 consecutivenucleotides selected from the nucleotide sequence between position 1 and1000 of SEQ ID NO: 25. The associated second primer of a primer paircomprises at least 15, at least 18, at least 20, at least 21 or at least24 consecutive nucleotides selected from the complementary nucleotidesequence between position 3314 and 2312 of SEQ ID NO: 25.

A person skilled in the art will find instructions and information onPCR for example in the handbook “PCR Strategies” (Innis, Felfand andSninsky, Academic Press, Inc., 1995), in the handbook by Diefenbach andDveksler “PCR Primer—a laboratory manual” (Cold Spring Harbor LaboratoryPress, 1995), in Gait's handbook “Oligonucleotide Synthesis: a PracticalApproach” (IRL Press, Oxford, UK, 1984) and in Newton and Graham “PCR”(Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).

Further instructions on PCR can be found for example in WO 06/100177 onpages 15 to 17.

In a further step, the nucleotide sequence of the polynucleotide is thendetermined. This can for example be determined by the chain-terminatingtechnique of Sanger et al. (Proceedings of the National Academies ofSciences, USA, 74, 5463-5467 (1977)) with the modifications stated byZimmermann et al. (Nucleic Acids Research 18, 1067 pp (1990)).

The polypeptide encoded by this nucleotide sequence can then be analyzedwith respect to the amino acid sequence. For this, the nucleotidesequence is input in a program for translating a DNA sequence into anamino acid sequence. Suitable programs are for example the “Patentin”program, which is obtainable from patent offices, for example the USPatent Office (USPTO), or the “Translate Tool”, which is available onthe ExPASy Proteomics Server on the World Wide Web (Gasteiger et al.,Nucleic Acids Research 31, 3784-3788 (2003)).

It is also possible for the polynucleotide, which is also designatedhereinafter as gltA allele, to be produced by methods of in-vitrogenetics.

Suitable methods for in-vitro mutagenesis including among otherstreatment with hydroxylamine according to Miller (Miller, J. H.: A ShortCourse in Bacterial Genetics. A Laboratory Manual and Handbook forEscherichia coli and Related Bacteria, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 1992) or the use of a polymerase chainreaction using a DNA polymerase, which has a high error rate. Such a DNApolymerase is for example the Mutazyme DNA Polymerase (GeneMorph PCRMutagenesis Kit, No. 600550) of the company Stratagene (La Jolla,Calif., USA). It is also possible to use mutagenic oligonucleotides, asdescribed by T. A. Brown (Gentechnologie für Einsteiger, SpektrumAkademischer Verlag, Heidelberg, 1993) and R. M. Horton (PCR-mediatedrecombination and mutagenesis, Molecular Biotechnology 3, 93-99 (1995)).The method using the “Quik Change Site-directed Mutagenesis Kit” of thecompany Stratagene (La Jolla, Calif., USA) described by Papworth et al.(Strategies 9(3), 3-4 (1996)) can also be used.

Methods for the determination of citrate synthase activity can be foundin Eikmanns et al. (Microbiology 140, 1817-1828 (1994)) and in Shiio etal. (Agricultural and Biological Chemistry 46(1), 101-107 (1982)).

It is moreover possible to overexpress the citrate synthase alleleaccording to the invention in Corynebacterium glutamicum or Escherichiacoli, and it can then be prepared in purified or isolated form.

A polynucleotide with the nucleotide sequence shown in SEQ ID NO: 5 wasisolated in this way. The polypeptide encoded by this polynucleotide isshown in SEQ ID NO: 6 and 8. It contains L-valine instead of L-asparticacid at position 5 of the amino acid sequence.

It was found that when the strain ATCC13032 is provided, instead of thewild-type gltA gene, with the gltA allele according to the invention,which codes for the citrate synthase according to SEQ ID NO: 6(ATCC13032::gltA D5V), in comparison with the wild-type strainATCC13032, which contains the citrate synthase according to SEQ ID NO:2, with enzyme activity reduced by approx. 40% up to a maximum ofapprox. 90%, preferably with enzyme activity reduced by approx. 70% upto a maximum of approx. 90%.

It is known that conservative amino acid substitutions only change theenzyme activity insignificantly. Accordingly, the invention also relatesto polynucleotides that code for polypeptides with citrate synthaseactivity, which in addition to the amino acid substitutions at position5 of the amino acid sequence contain one (1) or more conservative aminoacid substitution(s), which does not alter the enzyme activitysubstantially. That is, it remains essentially unchanged. The term “notaltered substantially,” “essentially unchanged,” or “substantiallyunchanged” means in this context that the citrate synthase activity ofthe polypeptide is altered by at most 20%, preferably at most 10% andmore preferably at most 5% to at most 2% in comparison with the citratesynthase activity of the polypeptide according to SEQ ID NO: 10 or SEQID NO: 6, preferably SEQ ID NO: 6.

For an experimental test, the gltA gene of strain ATCC13032 issubstituted for the gltA allele, which codes for a polypeptidecontaining the amino acid substitution at position 5 and at least oneconservative amino acid substitution. Then the strain is cultivated, acellular extract is produced and the citrate synthase activity isdetermined. As a reference, the citrate synthase activity in strainATCC13032::gltA D5V is determined.

Instead of strain ATCC13032, it is also possible to useL-lysine-excreting strains of Corynebacterium glutamicum which comprisesthe coding region of the gltA gene of the wild type including thenucleotide sequences located upstream, corresponding to the nucleotidesequence between position 1 and 2064 of SEQ ID NO: 3, preferably SEQ IDNO: 3. Suitable strains are, for example, DSM16833 described inPCT/EP2005/012417, DSM13994 described in EP 1 239 040 A2 or DSM17576described in DE 102005045301. In these strains, the appropriatemutation(s) can be inserted in the coding region of the gltA gene by,for example, allelic substitution.

It is also possible to purify the polypeptides and to conduct thecomparative tests on the purified polypeptides.

The enzyme citrate synthase (EC No. 4.1.3.7) catalyzes the condensationreaction of oxaloacetate and acetyl-CoA, with formation of citric acidand coenzyme A (CoA) as reaction products. The enzyme is assigned thenumber EC 2.3.3.1 in the Kyoto Encyclopedia of Genes and Genomes (KEGG,Kanehisa Laboratory, Bioinformatics Center, Institute for ChemicalResearch, Kyoto University, Japan).

In the case of aromatic L-amino acids, we talk of conservativesubstitutions when L-phenylalanine, L-tryptophan and L-tyrosine aresubstituted for one another. In the case of hydrophobic L-amino acids,we talk of conservative substitutions when L-leucine, L-isoleucine andL-valine are substituted for one another. In the case of polar L-aminoacids, we talk of conservative substitutions when L-glutamine andL-asparagine are substituted for one another. In the case of basicL-amino acids, we talk of conservative substitutions when L-arginine,L-lysine and L-histidine are substituted for one another. In the case ofacidic L-amino acids, we talk of conservative substitutions whenL-aspartic acid and L-glutamic acid are substituted for one another. Inthe case of L-amino acids containing hydroxyl groups, we talk ofconservative substitutions when L-serine and L-threonine are substitutedfor one another.

Preferably the polypeptide contains at most two (2), at most three (3),at most four (4) or at most five (5) conservative amino acidsubstitutions in addition to the substitution at position 5 of SEQ IDNO: 2.

It is known that the terminal methionine may be removed during proteinsynthesis by enzymes that are intrinsic to the host, so-calledaminopeptidases.

The isolated polynucleotides, which code for the citrate synthasevariant, or portions thereof, can be used for producing recombinantstrains of the genus Corynebacterium, preferably Corynebacteriumglutamicum, which comprises the amino acid substitution at position 5 ofthe amino acid sequence of the citrate synthase polypeptide and whichprovide improved release of L-amino acids into the surrounding medium oraccumulation of them inside the cell, compared with the starting orparent strain.

The initial strains preferably used are those which already possess thecapacity to excrete at least 1 g/l, preferably at least 5 g/l, and morepreferably at least 10 g/l of the desired L-amino acid into thesurrounding nutrient medium.

A widely used method for incorporating mutations in genes of bacteria ofthe genus Corynebacterium, preferably of the species Corynebacteriumglutamicum, is allelic substitution, which is also known as “genereplacement”. In this technique, a DNA fragment that contains themutation of interest is transferred into the desired strain and themutation is incorporated in the chromosome of the desired strain by atleast two recombination events or cross-over events or a gene sequencepresent in the strain in question is replaced by the mutated sequence.

In this method, the DNA fragment containing the mutation of interest maybe located in a vector, preferably a plasmid, which preferably is notreplicated by the strain that is to be provided with the mutation, orsuch replication is limited, i.e. occurs under selected cultureconditions. A bacterium of the genus Escherichia, preferably of thespecies Escherichia coli, may be used as auxiliary or intermediate host,in which the vector can be replicated.

Examples of such plasmid vectors are the pK*mob and pK*mobsacB vectors,for example pK18mobsacB, described by Schäfer et al. (Gene 145, 69-73(1994)), and the vectors described in WO 02/070685 and WO 03/014362.These vectors can replicate in Escherichia coli but not inCorynebacterium. Preferably, suitable vectors are those which contain agene with conditionally negative dominant action for example the sacBgene (levansucrase gene) of for example Bacillus or the galK gene(galactose kinase gene) of for example Escherichia coli. “Gene withconditionally negative dominant action” means a gene which under certainconditions is disadvantageous, for example toxic to the host, but inother conditions does not have adverse effects on the host carrying thegene. These make it possible to select for recombination events in whichthe vector is eliminated from the chromosome.

Furthermore, Nakamura et al. (U.S. Pat. No. 6,303,383) described atemperature-sensitive plasmid for Corynebacterium, which can onlyreplicate at temperatures below 31° C. It can also be used for thepurposes of the invention.

The vector is then transferred into the Corynebacterium by conjugation,for example, by Schäfer's method (Journal of Bacteriology 172, 1663-1666(1990)) or transformation, for example, by Dunican and Shivnan's method(Bio/Technology 7, 1067-1070 (1989)) or the method of Thierbach et al.(Applied Microbiology and Biotechnology 29, 356-362 (1988)). Optionally,the transfer of the DNA can also be achieved by ballistic methods (e.g.particle bombardment).

After homologous recombination by means of a first cross-over eventproducing integration and a suitable second cross-over event causing anexcision in the target gene or in the target sequence, incorporation ofthe mutation is achieved and a recombinant bacterium is obtained.“Target gene” means the gene in which the desired substitution is totake place.

The strains obtained can be identified and characterized using, amongothers, the methods of Southern blotting hybridization, polymerase chainreaction and sequencing, the method of fluorescence resonance energytransfer (FRET) (Lay et al. Clinical Chemistry 43, 2262-2267 (1997)) ormethods of enzymology.

This method was used by Schwarzer and Pühler (Bio/Technology 9, 84-87(1991)) for incorporating a lysA allele carrying a deletion, and a lysAallele carrying an insertion, into the chromosome of C. glutamicuminstead of the wild-type gene.

This method was used by Nakagawa et al. (EP 1108790) and Ohnishi et al.(Applied Microbiology and Biotechnology 58(2), 217-223 (2002)) forincorporating various mutations into the chromosome of C. glutamicumstarting from the isolated alleles or polynucleotides. In this way,Nakagawa et al. succeeded in incorporating a mutation designatedVal59Ala into the homoserine dehydrogenase gene (hom), a mutationdesignated Thr311Ile into the aspartate kinase gene (lysC or ask), amutation designated Pro458Ser into the pyruvate carboxylase gene (pyc)and a mutation designated Ala213Thr into theglucose-6-phosphate-dehydrogenase gene (zwf) of C. glutamicum strains.

For inserting the mutation in the gltA gene into the chromosome by meansof allelic substitution, it is possible to use a polynucleotide thatcodes for an amino acid sequence which has the amino acid substitutionat position 5 of SEQ ID NO: 2, as shown in SEQ ID NO: 10, and possesses,upstream and downstream thereof, a nucleotide sequence with a length ineach case of at least approx. 51 (cf. SEQ ID NO: 11 and 12) preferablyin each case at least approx. 101 or 102 (cf. SEQ ID NO: 13 and 14),preferably in each case at least approx. 201 nucleobases (cf. SEQ ID NO:15 and 16) and more preferably in each case at least approx. 500 or 498nucleobases (cf. SEQ ID NO: 17 and 18) selected from SEQ ID NO: 9. Themaximum length of the nucleotide sequence located upstream anddownstream of the mutation is generally approx. 500, approx. 750,approx. 1000, approx. 1500, approx. 2000 to 2100 nucleobases. Thenucleotide sequence located upstream of the mutation comprises, forexample, the sequence between position 1 to 762 of SEQ ID NO: 9 or thesequence between position 1 to 1012 of SEQ ID NO: 25. The nucleotidesequence located downstream of the mutation comprises for example thesequence between position 766 to 2814 of SEQ ID NO: 9 or the sequencebetween position 1016 to 3314 of SEQ ID NO: 25. The total length of thepolynucleotide used for the allelic substitution is accordingly at mostapprox. 1000, at most approx. 1500, at most approx. 2000, at mostapprox. 3000 or at most approx. 4000 to 4200 nucleobases.

Accordingly, the invention relates to a polynucleotide that comprises anucleotide sequence which contains, from position 1 to 39, thenucleotide sequence corresponding to position 1 to 39 of SEQ ID NO: 11and, from position 40 to 105, a nucleotide sequence that codes for theamino acid sequence according to SEQ ID NO: 12, having everyproteinogenic amino acid except L-aspartic acid being contained atposition 5.

In this context, the stated positions 1 to 39 of SEQ ID NO: 11correspond to the stated positions 712 to 750 of SEQ ID NO: 9. Thestated positions 40 to 105 of SEQ ID NO: 11 correspond to the statedpositions 751 to 816 of SEQ ID NO: 9.

In one embodiment, the polynucleotide comprises the nucleotide sequenceof SEQ ID NO: 11, with the codon corresponding to position 52 to 54coding for every proteinogenic amino acid except L-aspartic acid.

In another embodiment the polynucleotide comprises the nucleotidesequence from position 712 to 816 of SEQ ID NO: 7.

Accordingly, the invention also relates to a polynucleotide thatcomprises a nucleotide sequence which comprises, from position 1 to 89,the nucleotide sequence corresponding to position 1 to 89 of SEQ ID NO:13 and, from position 90 to 206, a nucleotide sequence which codes forthe amino acid sequence according to SEQ ID NO: 14, having everyproteinogenic amino acid except L-aspartic acid being contained atposition 5.

In this context, the stated positions 1 to 89 of SEQ ID NO: 13correspond to the stated positions 662 to 750 of SEQ ID NO: 9. Thestated positions 90 to 206 of SEQ ID NO: 13 correspond to the statedpositions 751 to 867 of SEQ ID NO: 9.

In one embodiment, the polynucleotide comprises the nucleotide sequenceof SEQ ID NO: 13, with the codon corresponding to position 102 to 104coding for every proteinogenic amino acid except L-aspartic acid.

In another embodiment, the polynucleotide comprises the nucleotidesequence from position 662 to 867 of SEQ ID NO: 7.

Accordingly, the invention also relates to a polynucleotide thatcomprises a nucleotide sequence which comprises, from position 1 to 189,the nucleotide sequence corresponding to position 1 to 189 of SEQ ID NO:15 and, from position 190 to 405, a nucleotide sequence which codes forthe amino acid sequence according to SEQ ID NO: 16, having everyproteinogenic amino acid except L-aspartic acid being contained atposition 5.

In this context, the stated positions 1 to 189 of SEQ ID NO: 15correspond to the stated positions 562 to 750 of SEQ ID NO: 9. Thestated positions 190 to 405 of SEQ ID NO: 15 correspond to the statedpositions 751 to 966 of SEQ ID NO: 9.

In one embodiment, the polynucleotide comprises the nucleotide sequenceof SEQ ID NO: 15, with the codon corresponding to position 202 to 204coding for every proteinogenic amino acid except L-aspartic acid.

In another embodiment, the polynucleotide comprises the nucleotidesequence from position 562 to 966 of SEQ ID NO: 7.

Accordingly, the invention also relates to a polynucleotide thatcomprises a nucleotide sequence which comprises, from position 1 to 488,the nucleotide sequence corresponding to position 1 to 488 of SEQ ID NO:17 and, from position 489 to 1001, a nucleotide sequence which codes forthe amino acid sequence according to SEQ ID NO: 18, with everyproteinogenic amino acid except L-aspartic acid being contained atposition 5.

In this context, the stated positions 1 to 488 of SEQ ID NO: 17correspond to the stated positions 263 to 750 of SEQ ID NO: 9. Thestated positions 489 to 1001 of SEQ ID NO: 15 correspond to the statedpositions 751 to 1263 of SEQ ID NO: 9.

In one embodiment, the polynucleotide comprises the nucleotide sequenceof SEQ ID NO: 17, with the codon corresponding to position 501 to 503coding for every proteinogenic amino acid except L-aspartic acid.

In another embodiment, the polynucleotide comprises the nucleotidesequence from position 263 to 1263 of SEQ ID NO: 7.

In another embodiment, the polynucleotide comprises the nucleotidesequence from position 9 to 1687 of SEQ ID NO: 31.

The invention also relates to vectors, preferably plasmids, comprisingthe stated polynucleotides.

The invention also relates to bacteria preferably of the genusEscherichia, more preferably of the species Escherichia coli, andCorynebacterium, more preferably of the species Corynebacteriumglutamicum, comprising the stated vectors.

The invention also relates to strains of the genus Corynebacterium,preferably of the species Corynebacterium glutamicum, which have beenproduced using the polynucleotides or vectors comprising thepolynucleotides.

It is also possible to insert the gltA allele at another site in thechromosome of Corynebacterium glutamicum. Possible examples are thesites or genes aecD, ccpA1, ccpA2, citA, citB, citE, fda, gluA, gluB,gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, menE, mqo, pck, pgi andpoxB, as described in WO 03/04037. Other possibilities are, for example,intergenic regions, DNA of prophages, defective phages and phagecomponents, as described in WO 04/069996.

The obtained recombinant strains display, relative to the initial strainor parent strain used, increased excretion or production of the desiredamino acid in a fermentation process.

The L-lysine-excreting starting strains that can be used for thepurposes of the invention possess, in addition to other properties, inparticular a lysine-insensitive aspartate kinase.

“Lysine-insensitive aspartate kinase” means a polypeptide or proteinwith aspartate kinase activity (EC No. 2.7.2.4), which in comparisonwith the wild form, have lower sensitivity to inhibition by mixtures oflysine and threonine or mixtures of AEC (aminoethylcysteine) andthreonine or lysine alone or AEC alone. Aspartate kinases of this kindare also called feedback-resistant or desensitized aspartate kinases.The nucleotide sequences coding for these desensitized aspartate kinasesor aspartate kinase variants are also designated as lysC^(FBR) alleles.Information on numerous lysC^(FBR) alleles is available in publicdatabases. The lysC-gene is also designated as the ask-gene by someauthors.

The coding region of the wild-type lysC gene of Corynebacteriumglutamicum corresponding to access number AX756575 of the NCBI databaseis shown in SEQ ID NO: 19 and the polypeptide encoded by this gene isshown in SEQ ID NO: 20. The nucleotide sequences located upstream of the5′ end and downstream of the 3′ end of the coding region are also shownin SEQ ID NO: 21. SEQ ID NO: 20 corresponds to SEQ ID NO: 22.

The L-lysine-excreting bacteria preferably have a lysC allele, whichcodes for an aspartate kinase variant possessing the amino acid sequenceof SEQ ID NO: 20, comprising one or more of the amino acid substitutionsselected from the group:

a) LysC A279T (substitution of L-alanine at position 279 of the encodedaspartate kinase protein according to SEQ ID NO: 20 for L-threonine; seeU.S. Pat. No. 5,688,671 and access numbers E06825, E06826, E08178 and174588 to 174597),

b) LysC A279V (substitution of L-alanine at position 279 of the encodedaspartate kinase protein according to SEQ ID NO: 20 for L-valine, see JP6-261766 and access number E08179),

c) LysC L297Q (substitution of L-leucine at position 297 of the encodedaspartate kinase protein according to SEQ ID NO: 20 for anotherproteinogenic amino acid, preferably L-glutamine; see DE 102006026328),

d) LysC S301F (substitution of L-serine at position 301 of the encodedaspartate kinase protein according to SEQ ID NO: 20 for L-phenylalanine;see U.S. Pat. No. 6,844,176 and access number E08180),

e) LysC S301Y (substitution of L-serine at position 301 of the encodedaspartate kinase protein according to SEQ ID NO: 20 for L-tyrosine, seeKalinowski et al. (Molecular and General Genetics 224, 317-324 (1990))and access number X57226),

f) LysC T308I (substitution of L-threonine at position 308 of theencoded aspartate kinase protein according to SEQ ID NO: 20 forL-isoleucine; see JP 6-261766 and access number E08181),

g) LysC T311I (substitution of L-threonine at position 311 of theencoded aspartate kinase protein according to SEQ ID NO: 20 forL-isoleucine; see WO 00/63388 and U.S. Pat. No. 6,893,848),

h) LysC S317A (substitution of L-serine at position 317 of the encodedaspartate kinase protein according to SEQ ID NO: 20 for L-alanine; seeU.S. Pat. No. 5,688,671 and access number 174589),

i) LysC R320G (substitution of L-arginine at position 320 of the encodedaspartate kinase protein according to SEQ ID NO: 20 for glycine; seeJetten et al. (Applied Microbiology and Biotechnology 43, 76-82 (995))and access number L27125),

j) LysC G345D (substitution of glycine at position 345 of the encodedaspartate kinase protein according to SEQ ID NO: 20 for L-aspartic acid;see Jetten et al. (Applied Microbiology and Biotechnology 43, 76-82(995)) and access number L16848),

k) LysC T380I (substitution of L-threonine at position 380 of theencoded aspartate kinase protein according to SEQ ID NO: 20 forL-isoleucine; see WO 01/49854 and access number AX192358), and

l) LysC S381F (substitution of L-serine at position 381 of the encodedaspartate kinase protein according to SEQ ID NO: 20 for L-phenylalanine;see EP 0435132).

Strains comprise aspartate kinase variants comprising the amino acidsubstitution LysC T311I or at least one amino acid substitution selectedfrom the group LysC A279T, LysC L297Q, LysC S317A, LysC T380I and LysCS381F.

Naturally it is also possible for insertion of the mutation in the gltAgene of the chromosome of a bacterium of the genus Corynebacterium to becarried out first, followed by insertion of one or more of the desiredmutation(s) in the lysC gene of the strain in question.

In one embodiment, the described aspartate kinases are overexpressed inthe Corynebacterium which comprises the amino acid substitutionaccording to the invention in the gltA gene.

Overexpression means an increase in the intracellular concentration oractivity of a ribonucleic acid, a protein or an enzyme compared with theinitial strain (parent strain) or wild-type strain. Initial strain(parent strain) means the strain on which the measure leading tooverexpression was carried out.

The increase in concentration or activity can be achieved, for example,by increasing the copy number of the corresponding polynucleotideschromosomally or extrachromosomally by at least one copy.

A widely used method of increasing the copy number comprises insertingthe corresponding polynucleotide in a vector, preferably a plasmid,which is replicated by a coryneform bacterium. Suitable plasmid vectorsare, for example, pZ1 (Menkel et al., Applied and EnvironmentalMicrobiology (1989) 64: 549-554) or the pSELF vectors described by Tauchet al. (Journal of Biotechnology 99, 79-91 (2002)). A review article onthe subject of plasmids in Corynebacterium glutamicum can be found inTauch et al. (Journal of Biotechnology 104, 27-40 (2003)).

Transposons, insertion elements (IS elements) or phages can also be usedas vectors. Such genetic systems are stated for example in patentspecifications U.S. Pat. No. 4,822,738, U.S. Pat. No. 5,804,414 and U.S.Pat. No. 5,804,414. Similarly, it is possible to use the IS elementISaB1 described in WO 92/02627 or the transposon Tn45 of plasmidpXZ10142 (cited in “Handbook of Corynebacterium glutamicum” (Publisher:L. Eggeling and M. Bott)).

Another widely used method for achieving overexpression is the techniqueof chromosomal gene amplification. In this method, at least oneadditional copy of the polynucleotide of interest is inserted in thechromosome of a coryneform bacterium. Such amplification techniques aredescribed for example in WO 03/014330 or WO 03/040373.

Another method of achieving overexpression comprises operably linkingthe corresponding gene or allele with a promoter or an expressioncassette. Suitable promoters for Corynebacterium glutamicum aredescribed for example in FIG. 1 of the review article by Patek et al.(Journal of Biotechnology 104(1-3), 311-323 (2003)). The variants of thedapA promoter described by Vasicova et al. (Journal of Bacteriology 181,6188-6191 (1999)), for example the promoter A25, can be used similarly.It is also possible to use the gap-promoter of Corynebacteriumglutamicum (EP 06007373). Finally it is possible to use the sufficientlywell-known promoters T3, T7, SP6, M13, lac, tac and trc described byAmann et al. (Gene 69(2), 301-315 (1988)) and Amann and Brosius (Gene40(2-3), 183-190 (1985)). Such a Such a promoter can for example beinserted upstream of the gene in question, typically at a distance ofabout 1-500 nucleobases from the start codon.

As a result of the measures for overexpression, the activity orconcentration of the corresponding polypeptide is increased by at least10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, at most up to1000% or 2000% relative to the activity or concentration of thepolypeptide in the strain prior to the measure leading tooverexpression.

In a further embodiment, the bacteria of the genus Corynebacterium,which preferably in addition comprises a polynucleotide that codes for alysine-insensitive aspartate kinase variant, possess one or more of thecharacters selected from the group

a) overexpressed polynucleotide (dapA gene), which codes for adihydrodipicolinate synthase (DapA, EC No. 4.2.1.52),

b) overexpressed polynucleotide (asd gene), which codes for an aspartatesemialdehyde dehydrogenase (Asd, EC No. 1.2.1.11),

c) overexpressed polynucleotide (lysA gene), which codes for adiaminopimelate decarboxylase (LysA, EC No. 4.1.1.20),

d) overexpressed polynucleotide (aat gene), which codes for an aspartateaminotransferase (Aat, EC No. 2.6.1.1),

e) overexpressed polynucleotide (lyse gene), which codes for apolypeptide with L-lysine exporting activity (LysE, Lysin EffluxPermease),

f) switched-off or attenuated activity of malate dehydrogenase (Mdh, ECNo. 1.1.1.37),

g) switched-off or attenuated activity of malate-quinone oxidoreductase(Mqo, EC No. 1.1.99.16),

h) overexpressed polynucleotide, which codes for a pyruvate carboxylase(Pyc, EC No. 6.4.1.1), and

i) switched-off or attenuated activity of the E1p subunit of thepyruvate dehydrogenase complex (AceE, EC No. 1.2.4.1).

Characters a) to g) are preferred.

The known genes, for example, the wild-type genes, of Escherichia coli(Blattner et al., Science 277(5), 1453-1462 (1997)), Bacillus subtilis(Kunst et al., Nature 390 (6657), 249-256 (1997)), Bacilluslicheniformis (Veith et al., Journal of Molecular Microbiology andBiotechnology 7 (4), 204-211 (2004)), Mycobacterium tuberculosis(Fleischmann et al., Journal of Bacteriology 1841, 5479-5490 (2004)),Mycobacterium bovis (Gamier et al., Proceedings of the National Academyof Sciences USA 100 (13), 7877-7882 (2003)), Streptomyces coeliclor(Redenbach et al., Molecular Microbiology 21 (1), 77-96 (1996)),Lactobacillus acidophilus (Altermann et al., Proceedings of the NationalAcademy of Sciences USA 102 (11), 3906-3912 (2005)), Lactobacillusjohnsonii (Pridmore et al., Proceedings of National Academy of SciencesUSA 101 (8), 2512-2517 (2004)), Bifidobacterium longum (Schell et al.,Proceedings of National Academy of Sciences USA 99 (22), 14422-14427(2002)), and Saccharomyces cerevisiae can be used for overexpression ofthe listed genes or polynucleotides. The genomes of the wild-type formsof these bacteria are available in sequenced or annotated form.Preferably the endogenous genes or polynucleotides of the genusCorynebacterium, more preferably of the species Corynebacteriumglutamicum, are used.

“Endogenous genes or polynucleotides” means the open reading frames(ORF), genes or alleles or their polynucleotides present in thepopulation of a species.

The dapA gene of Corynebacterium glutamicum strain ATCC13032 isdescribed for example in EP 0 197 335. The MC20 and MA16 mutations ofthe dapA promoter, as described in U.S. Pat. No. 6,861,246, can also beused, among others, for overexpression of the dapA gene ofCorynebacterium glutamicum.

The asd gene of Corynebacterium glutamicum strain ATCC21529 is describedfor example in U.S. Pat. No. 6,927,046.

The lysA gene of Corynebacterium glutamicum ATCC13869 (Brevibacteriumlactofermentum) is described for example in U.S. Pat. No. 6,090,597.

The aat gene of Corynebacterium glutamicum ATCC13032 is described forexample in Kalinowski et al. (Journal of Biotechnology 104 (1-3), 5-25(2003); see also access number NC_(—)006958). There it is designatedaspB gene. In U.S. Pat. No. 6,004,773 a gene coding for an aspartateaminotransferase is designated aspC. Marienhagen et al. (Journal ofBacteriology 187 (22), 7639-7646 (2005) denote the aat gene as aspTgene.

The lysE gene of Corynebacterium glutamicum R127 is described forexample in U.S. Pat. No. 6,858,406. Strain R127 is arestriction-defective mutant of ATCC13032 (Liebl et al., FEMSMicrobiology Letters 65, 299-304 (1989)). The lysE gene of strainATCC13032 used in U.S. Pat. No. 6,861,246 can be used similarly.

The pyc gene of Corynebacterium glutamicum of strain ATCC13032 isdescribed for example in WO 99/18228 and WO 00/39305. Furthermore,alleles of the pyc gene can be used, such as are described in U.S. Pat.No. 6,965,021. The pyruvate carboxylases described in this patentspecification possess one or more of the amino acid substitutionsselected from the group: Pyc E153D (substitution of L-glutamic acid atposition 153 for L-aspartic acid), Pyc A182S (substitution of L-alanineat position 182 for L-serine), Pyc A206S (substitution of L-alanine atposition 206 for L-serine), Pyc H227R (substitution of L-histidine atposition 227 for L-arginine), Pyc A455G (substitution of L-alanine atposition 455 for glycine), and Pyc D1120E (substitution of L-asparticacid at position 1120 for L-glutamic acid). Similarly, it is possible touse the pyc allele described in EP 1 108 790, which codes for a pyruvatecarboxylase containing the amino acid substitution Pyc P458S(substitution of L-proline at position 458 for L-serine).

“Switched-off or attenuated activity” means reduction or switching-offof the intracellular activity or concentration of one or more enzymes orproteins in a microorganism, which is encoded by the correspondingpolynucleotide or DNA.

For production of a strain in which the intracellular activity of adesired polypeptide is switched off, a deletion or insertion of at leastone (1) nucleobase, preferably of one (1) or of two (2) nucleobases, isinserted in the coding region of the corresponding gene. It is alsopossible to delete at least one (1) or more codon(s) within the codingregion. These measures lead to a shift of the reading frame (frame shiftmutations) and therefore typically to the synthesis of a nonfunctionalpolypeptide. The introduction of a nonsense mutation by transversion ortransition of at least one (1) nucleobase within the coding region has asimilar effect. Owing to the stop codon that forms, there is prematuretermination of translation. The stated measures are preferably carriedout in the region between the start codon and the penultimate codingcodon, more preferably in the 5′-terminal portion of the coding region,which codes for the N-terminus of the polypeptide. If the total lengthof a polypeptide (measured as the number of chemically bound L-aminoacids) is designated as 100%, then the portion of the amino acidsequence which, reckoned from the start amino acid L-formyl methionine,contains 80% of the subsequent L-amino acids, belongs to the N-terminusof the polypeptide.

Genetic measures for switching off malate-quinone oxidoreductase (Mqo)or reducing its expression are described for example in U.S. Pat. No.7,094,106. U.S. Pat. No. 7,094,106 describes switching off the mqo geneby incorporating deletions or insertions of at least one base pair orsubstitutions generating a stop codon into the mqo gene, whereinreduction of expression was achieved by placing the expression of themqo gene under the control of the E. coli trc promoter/LacI^(q)repressor system.

Genetic measures for switching off malate dehydrogenase (Mdh) aredescribed for example in WO 02/02778 (equivalent to U.S. Pat. No.6,995,002). In WO 02/02778, the mdh gene was switched off by theinsertion of a plasmid unable to replicate in Corynebacterium glutamicumcomprising a central part of the coding region of the mdh gene into thehost mdh gene by homologous recombination.

Genetic measures for switching off the E1p subunit (AceE) of thepyruvate dehydrogenase complex are described for example in EP 06119615and in Schreiner et al. (Journal of Bacteriology 187(17), 6005-6018(2005)). EP 06119615 and Schreiner et al. describe switching off theaceE gene by deleting a central part of the coding region of the aceEgene.

It is also possible, by suitable amino acid substitutions, to lower thecatalytic property of the polypeptide in question.

In the case of malate-quinone oxidoreductase (Mqo) this can be achieved,as described in WO 06/077004, by preparing or using alleles of the mqogene of SEQ ID NO: 23, which code for an Mqo variant that possesses theamino acid sequence of SEQ ID NO: 24 and contains one or more amino acidsubstitutions selected from the group

a) substitution of the L-serine at position 111 for anotherproteinogenic amino acid, preferably L-phenylalanine or L-alanine, and

b) substitution of the L-alanine at position 201 for anotherproteinogenic amino acid, preferably L-serine.

WO 06/077004 (equivalent to U.S. Pat. No. 7,214,526) describes anisolated coryneform bacterium mutant which comprises a gene encoding apolypeptide possessing malate quinone oxidoreductase enzyme activity,wherein the polypeptide comprises an amino acid sequence in which anyproteinogenic amino acid except L-serine is present at position 111 or acomparable position.

Strains that comprise an mqo allele that codes for an Mqo variant whichcomprises the amino acid sequence of SEQ ID NO: 24, and containsL-phenylalanine at position 111, are preferred.

By the measures of switching-off or attenuation, the activity orconcentration of the corresponding protein is generally lowered to 0 to75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity orconcentration of the wild-type protein, or of the activity orconcentration of the protein in the initial strain or parent strain.

The performance of the produced bacteria of the genus Corynebacterium orof the fermentation process using the produced bacteria with respect toone or more parameters selected from L-amino acid concentration (L-aminoacid formed per volume), L-amino acid yield (L-amino acid formed percarbon source consumed), L-amino acid formation (L-amino acid formed pervolume and time) and the specific L-amino acid formation (L-amino acidformed per cell dry mass or dry biomass and time or L-amino acid formedper cell protein and time) or other process variables and combinationsthereof, is increased by at least 0.5%, at least 1%, at least 1.5% or atleast 2% relative to the initial strain or parent strain or thefermentation process using them.

The produced bacteria of the genus Corynebacterium can be cultivatedcontinuously, as described for example in PCT/EP2004/008882, ordiscontinuously in a batch process, a fed batch process or a repeatedfed batch process, for the purpose of production of the desired L-aminoacids. A summary of a general nature covering known culture methods isgiven in Chmiel's textbook (Bioprozesstechnik 1. Einführung in dieBioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in thetextbook by Storhas (Bioreaktoren und periphere Einrichtungen (ViewegVerlag, Braunschweig/Wiesbaden, 1994)). PCT/EP2004/008882 (equivalent toWO 05/021772 and DE 10339847) describes a fermentation processcomprising incubating and culturing in at least first nutrient medium acoryneform bacterium producing L-lysine, feeding continuously furthernutrient media to the culture in one or several streams and removing atthe same time culturing broth with a removal stream or streamscorresponding to the feed streams, wherein over the entire period oftime of feeding and removing concentration of the source of carbon isnot more than 10 g/L and L-lysine is formed.

The culture medium or fermentation medium to be used matches therequirements of the particular strains. Descriptions of culture mediafor various microorganisms are given in “Manual of Methods for GeneralBacteriology” of the American Society for Bacteriology (Washington D.C.,USA, 1981). The terms culture medium and fermentation medium or mediumare interchangeable.

Sugars and carbohydrates, for example glucose, sucrose, lactose,fructose, maltose, molasses, sucrose-containing solutions from sugarbeet or sugar cane production, starch, hydrolyzed starch and cellulose,oils and fats, for example soya oil, sunflower oil, peanut oil andcoconut oil, fatty acids, for example palmitic acid, stearic acid andlinoleic acid, alcohols, for example glycerol, methanol and ethanol andorganic acids, for example acetic acid or lactic acid can be used as thecarbon source. These materials can be used individually or as a mixture.

Organic nitrogen-containing compounds such as peptones, yeast extract,meat extract, malt extract, corn-steep liquor, soybean flour and urea orinorganic compounds such as ammonium sulfate, ammonium chloride,ammonium phosphate, ammonium carbonate and ammonium nitrate can be usedas the nitrogen source. The nitrogen sources can be used individually oras a mixture.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used asthe phosphorus source.

The culture medium in addition contains salts, for example, in the formof chlorides or sulfates of metals such as sodium, potassium, magnesium,calcium and iron, for example magnesium sulfate or iron sulfate, whichare necessary for growth. Finally, essential growth substances can beused, such as amino acids for example homoserine and vitamins forexample thiamine, biotin or pantothenic acid, in addition to theaforementioned substances.

The aforementioned ingredients can be added to the culture as a singlecharge, or can be supplied in a suitable manner during cultivation.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammoniaor ammonia water, or acidic compounds such as phosphoric acid orsulfuric acid can be used in a suitable manner for pH control of theculture. The pH is generally adjusted to a value of 6.0 to 9.0,preferably 6.5 to 8. Antifoaming agents, for example polyglycol estersof fatty acids, can be used for controlling foaming. To maintain thestability of plasmids, suitable substances with selective action, forexample antibiotics, can be added to the medium. To maintain aerobicconditions, oxygen or oxygen-containing gas mixtures, for example air,are fed into the culture. The use of liquids enriched with hydrogenperoxide is also possible. Optionally, the fermentation is carried outat excess pressure, for example at a pressure of 0.03 to 0.2 MPa. Thetemperature of the culture is normally in the range from 20° C. to 45°C. and preferably from 25° C. to 40° C. In batch processes, cultivationis continued until a maximum of the desired L-amino acid has formed atgiven conditions. This goal is normally reached within 10 hours to 160hours. Longer cultivation times are possible with continuous processes.The activity of the bacteria leads to enrichment (accumulation) of theL-amino acid in the fermentation medium and/or in the bacterial cells.

Examples of suitable fermentation media are given inter alia in patentspecifications U.S. Pat. No. 5,770,409, U.S. Pat. No. 5,840,551 and U.S.Pat. No. 5,990,350 or U.S. Pat. No. 5,275,940.

Methods for the determination of L-amino acids are known. The analysiscan be carried out for example as described in Spackman et al.(Analytical Chemistry, 30, (1958), 1190) by anion-exchangechromatography followed by ninhydrin derivatization, or it can becarried out by reversed phase HPLC, as described by Lindroth et al.(Analytical Chemistry (1979) 51: 1167-1174).

Accordingly, the invention also relates to a method of production of anL-amino acid, wherein the following steps are carried out:

a) fermentation of the bacteria according to the invention in a suitablenutrient medium,

b) accumulation of the L-amino acid in the nutrient medium or in thecells of said bacteria.

These steps may be followed by collecting of the L-amino acid thataccumulated in the nutrient medium, in the fermentation broth or in thecells of the bacteria, in order to obtain a solid or a liquid product.

A fermentation broth means a fermentation medium or nutrient medium inwhich a microorganism has been cultivated for a certain time and at acertain temperature. The fermentation medium or the media used duringfermentation contain(s) all substances or components for ensuringmultiplication of the microorganism and formation of the desired aminoacid.

At the end of fermentation, the resulting fermentation broth accordinglycontains a) the biomass (cell mass) of the microorganism, formed as aresult of multiplication of the cells of the microorganism, b) thedesired L-amino acid that formed in the course of fermentation, such asL-lysine, L-valine or L-isoleucine, c) the organic by-products thatformed in the course of fermentation, and d) the constituents of thefermentation medium or of the ingredients for example vitamins such asbiotin, amino acids such as homoserine or salts such as magnesiumsulfate, that were not consumed in the fermentation.

The organic by-products include substances which may be produced and maybe excreted by the microorganisms used in the fermentation in additionto the particular desired organic compound. These also include sugars,for example trehalose.

In the case of the amino acids L-valine and L-isoleucine, isolation andpurification, for example using one or more methods selected from thegroup comprising chromatographic techniques, crystallization techniquesand the use of activated charcoal, is preferred, so that pure productsare largely obtained, for example products with purity of ≧90 wt. % or≧95 wt. %.

In the case of the amino acid L-lysine, essentially four differentproduct forms are known.

One group of L-lysine-containing products comprises concentrated,aqueous, alkaline solutions of purified L-lysine (EP-B-0534865). Anothergroup, as described for example in U.S. Pat. No. 6,340,486 and U.S. Pat.No. 6,465,025, comprises aqueous, acidic, biomass-containingconcentrates of L-lysine-containing fermentation broths. Another groupof solid products comprises powder or crystalline forms of purified orpure L-lysine, which may bey in the form of a salt, for example L-lysinemonohydrochloride. Yet another group of solid product forms is describedfor example in EP-B-0533039. The product form described there contains,in addition to L-lysine, most of the ingredients employed duringfermentation but not consumed, and possibly the biomass of themicroorganism used at a proportion of >0%-100%.

In accordance with the various product forms, a great variety of methodsis known for collecting, isolating or purifying the L-lysine from thefermentation broth, in order to produce an L-lysine-containing productor purified L-lysine.

Solid, pure L-lysine may be produced using methods of ion-exchangechromatography possibly with the use of activated charcoal andcrystallization techniques. In this way we obtain the corresponding baseor a corresponding salt, for example the monohydrochloride (Lys-HCl) orlysine sulfate (Lys₂-H₂SO₄).

A method of production of aqueous, basic L-lysine-containing solutionsfrom fermentation broths is described in EP-B-0534865. In the methoddescribed there, the biomass is separated from the fermentation brothand discarded. A pH between 9 and 11 is established by means of a base,for example, sodium, potassium or ammonium hydroxide. The mineralconstituents (inorganic salts) are separated from the broth afterconcentration and cooling, and either used as fertilizers or discarded.

In the case of methods for production of lysine using the bacteria,methods are preferred in which products are obtained that contain theconstituents of the fermentation broth. These are used in particular asanimal feed additives.

Depending on what is required, the biomass can be removed from thefermentation broth completely or partially by separation techniques suchas centrifugation, filtration, decanting or a combination thereof, or itcan be left in it completely. Optionally, the biomass or thefermentation broth containing the biomass is inactivated during asuitable process step, for example, by thermal treatment (heating) or byadding acid.

In one embodiment, the biomass is removed completely or almostcompletely, so that the finished product has a biomass content of zero(0%) or max. 30%, max. 20%, max. 10%, max. 5%, max. 1% or max. 0.1%. Inanother embodiment, the biomass is not removed or only a smallproportion is removed, so that the finished product contains all thebiomass (100%) or more than 70%, 80%, 90%, 95%, 99% or 99.9% biomass. Ina method according to the invention, the biomass is accordingly removedin proportions from ≧0% to ≦100%.

Finally, the fermentation broth obtained after the fermentation can beadjusted to an acid pH with an inorganic acid such as hydrochloric acid,sulfuric acid or phosphoric acid or organic acid such as propionic acid,before or after complete or partial removal of the biomass (GB 1,439,728or EP 1 331 220). It is also possible to acidify the fermentation brothstill containing all the biomass. Finally, the broth can also bestabilized by adding sodium bisulfite (NaHSO₃, GB 1,439,728) or anothersalt for example ammonium, alkali or alkaline-earth salt of sulfurousacid.

In separating the biomass, any organic or inorganic solids contained inthe fermentation broth are removed partially or completely. The organicby-products dissolved in the fermentation broth and the dissolved,unconsumed constituents of the fermentation medium (ingredients) remainin the product at least partially (>0%), preferably to at least 25%,preferably to at least 50% and more preferably to at least 75%.Optionally, these also remain in the product completely (100%) or almostcompletely, i.e. >95% or >98% or over 99%. If a product in this sensecontains at least a proportion of the constituents of the fermentationbroth, it is also described with the term “product based on fermentationbroth”.

Then the broth is dewatered or thickened or concentrated using knownmethods, e.g. by means of a rotary evaporator, thin-film evaporator,falling-film evaporator, by reverse osmosis or by nanofiltration. Thisconcentrated fermentation broth can then be processed by techniques offreeze drying, spray drying, spray granulation or by other methods, forexample the circulating fluidized bed as described in PCT/EP2004/006655,to pourable products and preferably to a fine powder or preferablycoarse granules. If required, a desired product can be isolated from thegranules thus obtained by sieving or dust separation.

It is also possible for the fermentation broth to be dried directly,i.e. without previous concentration, by spray drying or spraygranulation.

“Pourable” means powders which, from a series of glass discharge vesselswith outlet openings of different sizes, are discharged freely from thevessel with the 5 mm (millimeter) opening (Klein: Seifen, Öle, Fette,Wachse 94, 12 1968)).

“Fine” means a powder having mainly (>50%) a grain size of 20 to 200 μmdiameter. “Coarse” means a product having mainly (>50%) a grain sizefrom 200 to 2000 μm diameter.

Grain size can be determined using methods of laser diffractionspectrometry. The relevant methods are described in the textbook on“Particle Size Measurement in Laboratory Practice” by R. H. Müller andR. Schuhmann, Wissenschaftliche Verlagsgesellschaft Stuttgart (1996) orin the textbook “Introduction to Particle Technology” by M. Rhodes,Publ. Wiley & Sons (1998).

The pourable, fine powder can be converted by suitable compaction orgranulation techniques to a coarse, storable and largely dust-freeproduct with good pourability.

The term “dust-free” means that the product only contains a smallproportion (<5%) of particles with grain size under 100 μm diameter.

“Storable”, in the sense of this invention, means a product that can bestored in cool, dry conditions for at least one (1) year or longer,preferably at least 1.5 years or longer, more preferably two (2) yearsor longer, without any substantial loss (max. 5%) of the particularamino acid.

The invention further relates to a method of manufacturing an L-lysinecomprising product, which is described in broad outline in DE102006016158, and in which the fermentation broth obtained using themicroorganisms according to the invention, from which the biomass hasbeen optionally separated completely or partially, is further processed,by carrying out a process that comprises at least the following steps:

a) the pH is lowered to 4.0-5.2, preferably 4.9-5.1, by adding sulfuricacid, and a sulfate/L-lysine molar ratio of 0.85-1.2, preferably0.9-1.0, more preferably >0.9 to <0.95, is established in the broth, ifnecessary by adding one or more additional sulfate-containingcompound(s) and

b) the mixture thus obtained is concentrated by dewatering, andoptionally granulated,

optionally with one or both of the following measures being carried outbefore step a):

c) measurement of the sulfate/L-lysine molar ratio for determining therequired amount of sulfate-containing compound(s)

d) addition of a sulfate-containing compound selected from the groupcomprising ammonium sulfate, ammonium hydrogensulfate and sulfuric acidin suitable proportions.

Optionally, also prior to step b), a salt of sulfurous acid, preferablyan alkali metal hydrogensulfite, and more preferably sodiumhydrogensulfite, at a concentration of 0.01-0.5 wt. %, preferably0.1-0.3 wt. %, more preferably 0.1-0.2 wt. % relative to thefermentation broth is used.

DE 102006016158 (equivalent to US2007082031 and WO 07/042363) describesrelatively light and thermally stable granulatedfermentation-broth-based animal feed additives having a high content ofL-lysine and low-loss methods for production the additives from brothsobtained by fermentation.

As preferred sulfate-containing compounds in the sense of theaforementioned process steps we may mention in particular ammoniumsulfate and/or ammonium hydrogensulfate or corresponding mixtures ofammonia and sulfuric acid and sulfuric acid itself.

The sulfate/L-lysine molar ratio V is calculated from the formula:V=2×[SO₄ ²⁻]/[L-lysine]. This formula takes account of the fact that theSO₄ ²⁻ anion, or sulfuric acid, is divalent. A ratio V=1 means that theLys₂-H₂SO₄ is of stoichiometric composition, whereas at a ratio of V=0.9there is a 10% sulfate deficit and at a ratio of V=1.1 there is a 10%sulfate excess.

During granulating or compacting the usual organic or inorganicauxiliaries, or carriers such as starch, gelatin, cellulose derivativesor similar substances, as are usually employed in the processing offoodstuffs or animal feed as binders, gelling agents or thickeners, orother substances for example silicic acids, silicates (EP0743016A) orstearates may be used.

Treatment the surface of the obtained granules with oils, as describedin WO 04/054381 may be used. The oils used can be mineral oils,vegetable oils or mixtures of vegetable oils. Examples of such oils aresoya oil, olive oil, soya oil/lecithin mixtures. Similarly, siliconeoils, polyethylene glycols or hydroxyethyl cellulose are also suitable.Treatment of the surfaces with the aforesaid oils gives increasedabrasion resistance of the product and a reduction in the proportion ofdust. The content of oil in the product is 0.02-2.0 wt. %, preferably0.02-1.0 wt. %, and more preferably 0.2-1.0 wt. % relative to the totalamount of the feed additive.

Products are preferred having a proportion of ≧97 wt. % of a grain sizefrom 100 to 1800 μm or a proportion of ≧95 wt. % of a grain size from300 to 1800 μm diameter. The proportion of dust, i.e. particles with agrain size<100 μm, is preferably >0 to 1 wt. %, max. 0.5 wt. % beingmore preferred.

Alternatively, the product can also be coated with an organic orinorganic carrier that is known and usual in animal feed processing, forexample silicic acids, silicates, grits, bran, meal or flour, starch,sugars or other substances and/or mixed and stabilized with usualthickeners or binders. Examples of applications and the methods employedare described in the literature (Die Mühle+Mischfuttertechnik 132 (1995)49, page 817).

Finally, using coating processes with film-forming agents such as metalcarbonates, silicic acids, silicates, alginates, stearates, starches,gums and cellulose ethers, as described in DE-C-4100920, the product canbe brought to a state in which it is stable against digestion in animalstomachs preferably the stomach of ruminants. DE4100920 (equivalent toU.S. Pat. No. 5,279,832 and EP 0495349) describes an active-substancepreparation for oral administration, preferably for ruminants,comprising an active-substance core comprising at least one biologicallyactive substance and a coating around this core which delays the releaseof the core after oral administration due to its geometrical shape aswell as a method of preparing an accordingly shaped core pellet bycoating.

For establishing a desired L-lysine concentration in the product,depending on requirements, the L-lysine can be added during the processin the form of a concentrate or optionally a substantially puresubstance or salt thereof in liquid or solid form. These can be addedindividually or as mixtures to the fermentation broth obtained orconcentrated, or alternatively during the drying or granulation process.

The invention relates further to a method of production of a solidlysine-containing product, as described in broad outline in US20050220933, and which comprises the processing of the fermentationbroth obtained using the microorganisms according to the invention, inthe following steps:

a) filtration of the fermentation broth, preferably with a membranefilter, so that a biomass-containing sludge and a filtrate are obtained,

b) concentration of the filtrate, preferably so that a solids content of48-52 wt. % is obtained,

c) granulation of the concentrate obtained in step b), preferably at atemperature from 50° C. to 62° C., and

d) coating of the granules obtained in c) with one or more coatingagent(s)

For the coating in step d), it is preferable to use coating agents thatare selected from the group comprising

d1) the biomass obtained in step a)

d2) a compound containing L-lysine, preferably selected from the groupcomprising L-lysine hydrochloride or L-lysine sulfate,

d3) an essentially L-lysine-free material with L-lysine content<1 wt. %,preferably <0.5 wt. %, preferably selected from the group consisting ofstarch, carrageenan, agar, silicic acids, silicates, grits, bran andmeal, and

d4) a water-repellent substance, preferably selected from the groupconsisting of oils, polyethylene glycols and liquid paraffins.

The content of L-lysine is adjusted to a desired value by the measurescorresponding to steps d1) to d4), preferably d1) to d3).

In the production of L-lysine-containing products, the ratio of the ionsis preferably adjusted so that the molar ionic ratio according to thefollowing formula2×[SO₄ ²⁻]+[Cl⁻]—[NH₄ ⁺]—[Na⁻]—[K⁺]-2×[Mg²⁺]−2×[Ca²⁺]/[L-Lys] is0.68-0.95,preferably 0.68-0.90, as described by Kushiki et al. in US 20030152633.

In the case of lysine, the solid product based on fermentation brothproduced in this way has a lysine content (lysine base) from 10 wt. % to70 wt. % or 20 wt. % to 70 wt. %, preferably 30 wt. % to 70 wt. % andmore preferably from 40 wt. % to 70 wt. % based on the dry mass of theproduct. Maximum contents of lysine base of 71 wt. %, 72 wt. %, 73 wt. %are also possible. The water content of the L-lysine-containing, solidproduct is up to 5 wt. %, preferably up to 4 wt. %, and more preferablyless than 3 wt. %. Having generally described this invention, a furtherunderstanding can be obtained by reference to certain specific exampleswhich are provided herein for purposes of illustration only, and are notintended to be limiting unless otherwise specified.

EXAMPLES Example 1 Sequencing of the gltA Gene of the Strain DM678

The strain Corynebacterium glutamicum DM678 (U.S. Pat. No. 6,861,246) isa lysine-producing strain developed by mutagenesis and screening. It isauxotrophic for L-threonine and L-methionine-sensitive. The strain wasdeposited at the Deutsche Sammlung für Mikroorganismen und Zellkulturen(DSMZ, Braunschweig, Germany) as DSM12866.

The method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) wasused to isolate chromosomal DNA from the strain DM678. The polymerasechain reaction was used to amplify a DNA segment which harbors the gltAgene. The following oligonucleotides were used as primers for this:

gltA_XL-A1 (SEQ ID NO: 27): 5′ tgagttctattggcgtgacc 3′ gltA_XL-E1 (SEQID NO: 28): 5′ ttcgccaacgatgatgtcag 3′

The depicted primers were synthesized by MWG Biotech (Ebersberg,Germany). They make it possible to amplify a DNA segment which was about1.8 kb long and harbors the gltA gene. The primer gltA_XL-A1 binds tothe region corresponding to position 490 to 509 of the strandcomplementary to SEQ ID NO: 3 (and SEQ ID NO: 7). The primer gltA_XL-E1binds to the region corresponding to position 2266 to 2247 of the strandshown in SEQ ID NO: 3 (and SEQ ID NO: 7).

The PCR reaction was carried out using the Phusion high fidelity DNApolymerase (New England Biolabs, Frankfurt, Germany). The reactionmixture was made up as specified by the manufacturer and contained 10 μlof the 5× Phusion HF buffer supplied, deoxynucleoside triphosphates eachin a concentration of 200 μM, primers in a concentration of 0.5 μM,approximately 50 ng of template DNA and 2 units of Phusion polymerase ina total volume of 50 μl. The volume was adjusted to 50 μl by adding H₂O.

The PCR mixture was first subjected to an initial denaturation at 98° C.for 30 seconds. This was followed by a denaturation step at 98° C. for20 seconds, repeated 35×, a step for binding the primers to theintroduced DNA at 60° C. for 20 seconds and the extension step to extendthe primers at 72° C. for 60 seconds. After the final extension step at72° C. for 5 minutes, the PCR mixture was subjected to an agarose gelelectrophoresis (0.85% agarose). A DNA fragment about 1.8 kb long wasidentified, isolated from the gel and purified using the QIAquick gelextraction kit from Qiagen (Hilden, Germany).

The nucleotide sequence of the amplified DNA fragment or PCR product wasdetermined by Agowa (Berlin, Germany).

The nucleotide sequence of the coding region of the gltA allele from thestrain DM678 contains thymine as nucleobase at position 14. Thewild-type gene (see SEQ ID NO: 1) contains adenine as nucleobase at thisposition. This adenine-thymine transversion leads to an amino acidexchange from aspartate to valine at position 5 of the resulting aminoacid sequence. This mutation is referred to hereinafter as gltAD5V. Theallele gltAD5V is depicted in SEQ ID NO: 5, and the amino acid sequenceof the protein which was revealed with the aid of the Patentin programis depicted in SEQ ID NO: 6.

Example 2 Construction of the Exchange Vector pK18mobsacB_gltAD5V

The polymerase chain reaction was used to amplify a DNA fragment whichharbors part of the upstream region of the gltA gene and part of thecoding region which contains the gltAD5V mutation. The chromosomal DNAobtained in example 1 from DM678 was used as template. The followingoligonucleotides were selected as primers for the PCR:

gltA_1.p (SEQ ID NO: 29): 5′ CCGTCGACAATAGCCTGAA 3′ gltA_2.p (SEQ ID NO:30): 5′ CC-GAATTC-TTCGAGCATCTCCAGAAC 3′

They were synthesized by MWG Biotech (Ebersberg, Germany) and make itpossible to amplify a DNA segment about 1.7 kb long comprising 832 bp ofthe upstream region and nucleotides 1-855 bp of the coding region of thegltA gene from DM678.

The primer gltA_(—)1.p binds to the region corresponding to position 169to 187 of the strand complementary to SEQ ID NO: 25. Nucleotides 9 to 26of the primer gltA_(—)2.p bind to the region corresponding to position1855 to 1838 of the strand shown in SEQ ID NO: 25. In addition, theprimer gltA_(—)1.p contains the native cleavage site of the restrictionenzyme SalI, and the primer gltA_(—)2.p contains the sequence of thecleavage site of the restriction enzyme EcoRI, which are each marked byunderlining in the nucleotide sequence depicted above.

The PCR reaction was carried out using the Phusion high fidelity DNApolymerase (New England Biolabs, Frankfurt, Germany). The reactionmixture had the composition described above. The PCR was carried out asdescribed above. The nucleotide sequence of the amplicon about 1.7 kblong is depicted in SEQ ID NO: 31.

The amplicon was treated with the restriction endonucleases SalI andEcoRI and identified by electrophoresis in a 0.8% agarose gel. It wassubsequently isolated from the gel and purified using the QIAquick gelextraction kit from Qiagen.

The DNA fragment purified in this way contains the described gltAD5Vmutation and has ends compatible with DNA cut with SalI and EcoRI(respectively gltAD5V fragment and gltA′ in the FIGURE). It wassubsequently cloned into the mobilizable vector pK18mobsacB described bySchäfer et al. (Gene, 145, 69-73 (1994)) in order to make an allelic ormutation substitution possible. For this purpose, pK18mobsacB wasdigested with the restriction enzymes EcoRI and SalI, and the ends weredephosphorylated with alkaline phosphatase (alkaline phosphatase,Boehringer Mannheim, Germany). The vector prepared in this way was mixedwith the gltAD5V fragment, and the mixture was treated with theready-to-go T4 DNA ligase kit (Amersham-Pharmacia, Freiburg, Germany).

Subsequently, the E. coli strain S17-1 (Simon et al., Bio/Technologie 1:784-791, 1993) was transformed with the ligation mixture (Hanahan, In.DNA cloning. A practical approach. Vol. 1. ILR-Press, Cold SpringHarbor, N.Y., 1989). Selection of plasmid-harboring cells took place byplating out the transformation mixture on LB agar (Sambrook et al.,Molecular Cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor,N.Y., 1989) which had been supplemented with 25 mg/l kanamycin.

Plasmid DNA was isolated from a transformant using the QIAprep spinminiprep kit from Qiagen and checked by restriction cleavage with theenzymes SalI and EcoRI and subsequent agarose gel electrophoresis. Theplasmid was called pK18mobsacB_gltAD5V and is depicted in the FIGURE.The abbreviations and designations used have the following meaning. Thestated numbers of base pairs are approximations obtained within thescope of the reproducibility of measurements.

Kan: kanamycin-resistance gene SalI: cleavage site of the restrictionenzyme SalI EcoRI: cleavage site of the restriction enzyme EcoRI gltA′:cloned DNA fragment containing the gltAD5V mutation sacB: sacB geneRP4-mob: mob region with the origin of replication for transfer (oriT)oriV: origin of replication V

Example 3 Incorporation of the gltAD5V Mutation into the StrainCorynebacterium glutamicum DM1797

The intention was to introduce the gltAD5V mutation into the strainCorynebacterium glutamicum DM1797. The strain DM1797 was anaminoethylcysteine-resistant mutant of Corynebacterium glutamicumATCC13032 and described in PCT/EP/2005/012417. It was deposited underthe number DSM16833 at the Deutsche Sammlung für Mikroorganismen undZellkulturen (DSMZ, Braunschweig, Germany).

The vector pK18mobsacB_gltAD5V described in example 2 was transferred byconjugation according to the protocol of Schäfer et al. (Journal ofMicrobiology 172: 1663-1666 (1990)) into the C. glutamicum strainDM1797. The vector was incapable of independent replication in DM1797and was retained in the cell only if it was integrated into thechromosome as the result of a recombination event. Selection oftransconjugants, i.e. of clones having integrated pK18mobsacB_gltAD5V,took place by plating out the conjugation mixture on LB agar which hadbeen supplemented with 25 mg/l kanamycin and 50 mg/l of nalidixic acid.Kanamycin-resistant transconjugants were subsequently streaked onto LBagar plates supplemented with kanamycin (25 mg/l) and incubated at 33°C. for 24 hours. Mutants in which, as a result of a second recombinationevent, excision of the plasmid had taken place were selected byculturing the clones nonselectively in LB liquid medium for 30 hours,then streaking onto LB agar, which had been supplemented with 10%sucrose, and incubating at 33° C. for 24 hours.

The plasmid pK18mobsacB_gltAD5V contains, just like the initial plasmidpK18mobsacB, besides the kanamycin-resistance gene a copy of the sacBgene which codes for the levan sucrase from Bacillus subtilis. Thesucrose-inducible expression of the sacB gene leads to the formation oflevan sucrase which catalyzes the synthesis of the product levan whichis toxic for C. glutamicum. Thus, the only clones to grow onsucrose-supplemented LB agar were those in which the integratedpK18mobsacB_gltAD5V has been excised as the result of a secondrecombination event. Depending on the position of the secondrecombination event in relation to the site of mutation, the excision isassociated with allelic substitution or incorporation of the mutationinstead, or the original copy remains in the host's chromosome.

A clone in which the desired exchange, i.e. the incorporation of thegltAD5V mutation, had taken place was then sought. For this purpose, thesequence of the gltA gene was determined for 10 clones with thephenotype “growth in the presence of sucrose” and “non-growth in thepresence of kanamycin”. In this way, a clone harboring the gltAD5Vmutation was identified. This strain was designated C. glutamicumDM1797_gltAD5V.

Example 4 Production of L-lysine

The strain DM1797_gltAD5V obtained in example 3 and the initial strainDM1797 were cultured in a nutrient medium suitable for producing lysine,and the lysine content in the culture supernatant was determined.

For this purpose, the clones were initially grown on brain-heart agarplates (Merck, Darmstadt, Germany) at 33° C. for 24 hours. These agarplate cultures were each used for inoculation of a preculture (10 ml ofmedium in a 100 ml Erlenmeyer flask). The medium MM was used as mediumfor the precultures. The precultures were incubated at 33° C. and 240rpm on a shaker for 24 hours. Each of these precultures was used toinoculate a main culture, so that the initial OD (660 nm) of the mainculture was 0.1 OD. The medium MM was likewise used for the maincultures.

Medium MM CSL  5 g/l MOPS 20 g/l Glucose (autoclaved separately) 50 g/l

Salts:

(NH₄)₂SO₄) 25 g/l KH₂PO₄ 0.1 g/l MgSO₄ * 7 H₂O 1.0 g/l CaCl₂ * 2 H₂O 10mg/l FeSO₄ * 7 H₂O 10 mg/l MnSO₄ * H₂O 5.0 mg/l Biotin (sterilized byfiltration) 0.3 mg/l Thiamine * HCl (sterilized by filtration) 0.2 mg/lCaCO₃ 25 g/l

CSL (corn steep liquor), MOPS (morpholinopropanesulfonic acid) and thesalt solution were adjusted to pH 7 with aqueous ammonia and autoclaved.The sterile substrate and vitamin solutions, and the dry autoclavedCaCO₃, were then added.

Culturing took place in volumes of 10 ml which were present in 100 mlErlenmeyer flasks with baffles. The temperature was at 33° C., thenumber of revolutions was 250 rpm and the humidity was 80%.

After 48 hours, the optical density (OD) was determined at a measurementwavelength of 660 nm using a Biomek 1000 (Beckmann Instruments GmbH,Munich). The amount of lysine formed was determined using an amino acidanalyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion-exchangechromatography and post-column derivatization with ninhydrin detection.

TABLE 1 Strain OD (660) Lysine HCl (g/l) DM1797 11.9 4.8 DM1797_gltAD5V11.2 5.3

Example 5 Incorporation of the gltAD5V Mutation into the StrainBrevibacterium lactofermentum FERM BP-1763

It was intended to introduce the gltAD5V mutation into the strainBrevibacterium lactofermentum FERM BP-1763. The strain FERM BP-1763 is amycophenolic acid-resistant valine producer (U.S. Pat. No. 5,188,948).It is auxotrophic for L-isoleucine and L-methionine.

The vector pK18mobsacB_gltAD5V described in example 2 was transferred byconjugation according to the protocol of Schäfer et al. (Journal ofMicrobiology 172: 1663-1666 (1990)) into the strain FERM-BP-1763. Thevector was incapable of independent replication in FERM BP-1763 and wasretained in the cell only if it was integrated into the chromosome asthe result of a recombination event. Selection of transconjugants, i.e.of clones having integrated pK18mobsacB_gltAD5V, took place by platingout the conjugation mixture on LB agar which had been supplemented with25 mg/l kanamycin and 50 mg/l of nalidixic acid. Kanamycin-resistanttransconjugants were subsequently streaked onto LB agar platessupplemented with kanamycin (25 mg/l) and incubated at 33° C. for 24hours. Mutants in which, as a result of a second recombination event,excision of the plasmid had taken place were selected by culturing theclones nonselectively in LB liquid medium for 30 hours, then streakingonto LB agar, which had been supplemented with 10% sucrose, andincubating at 33° C. for 24 hours.

The plasmid pK18mobsacB_gltAD5V contained, just like the initial plasmidpK18mobsacB, besides the kanamycin-resistance gene a copy of the sacBgene which coded for the levan sucrase from Bacillus subtilis. Thesucrose-inducible expression of the sacB gene led to the formation oflevan sucrase which catalyzes the synthesis of the product levan whichwas toxic for C. glutamicum. Thus, the only clones to grow onsucrose-supplemented LB agar were those in which the integratedpK18mobsacB_gltAD5V has been excised as the result of a secondrecombination event. Depending on the position of the secondrecombination event in relation to the site of mutation, the excisionwas associated with allelic substitution or incorporation of themutation instead, or the original copy remains in the host's chromosome.

A clone in which the desired exchange, i.e. the incorporation of thegltAD5V mutation, had taken place was then sought. For this purpose, thesequence of the gltA gene was determined for 10 clones with thephenotype “growth in the presence of sucrose” and “non-growth in thepresence of kanamycin”. In this way, a clone harboring the gltAD5Vmutation was identified. This strain was designated C. glutamicum FERMBP-1763_gltAD5V.

Example 6 Production of L-valine

The strain FERM BP-1763_gltAD5V obtained in example 5 and the initialstrain FERM BP-1763 were cultured in a nutrient medium suitable forproducing valine, and the valine content in the culture supernatant wasdetermined.

For this purpose, the clones were initially grown on brain-heart agarplates (Merck, Darmstadt, Germany) at 33° C. for 24 hours. These agarplate cultures were each used for inoculation of a preculture (10 ml ofmedium in a 100 ml Erlenmeyer flask).

The medium CgIII (2.5 g/l NaCl, 10 g/l Bacto peptone, 10 g/l Bacto yeastextract, pH 7.4, 20 g/l glucose (autoclaved separately) was used for theprecultures. The precultures were incubated at 33° C. and 240 rpm on ashaker for 24 hours. Each of these precultures was used to inoculate amain culture, so that the initial OD (660 nm) of the main culture was0.1 OD. The medium MM was likewise used for the main cultures.

Medium MM CSL  5 g/l MOPS 20 g/l Glucose (autoclaved separately) 50 g/l

Salts:

(NH₄)₂SO₄) 25 g/l KH₂PO₄ 0.1 g/l MgSO₄ * 7 H₂O 1.0 g/l CaCl₂ * 2 H₂O 10mg/l FeSO₄ * 7 H₂O 10 mg/l MnSO₄ * H₂O 5.0 mg/l Isoleucine (sterilizedby filtration) 0.1 g/l Methionine (sterilized by filtration) 0.1 g/lLeucine (sterilized by filtration) 0.1 g/l Thiamine * HCl (sterilized byfiltration) 0.2 mg/l CaCO₃ 25 g/l

CSL (corn steep liquor), MOPS (morpholinopropanesulfonic acid) and thesalt solution were adjusted to pH 7 with aqueous ammonia and autoclaved.The sterile substrate and vitamin solutions, and the dry autoclavedCaCO₃, were then added.

Culturing took place in volumes of 10 ml which were present in 100 mlErlenmeyer flasks with baffles. The temperature was at 33° C., thenumber of revolutions was 250 rpm and the humidity was 80%.

After 48 hours, the optical density (OD) was determined at a measurementwavelength of 660 nm using the Biomek 1000 (Beckmann Instruments GmbH,Munich). The amount of valine formed was determined using an amino acidanalyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion-exchangechromatography and post-column derivatization with ninhydrin detection.

TABLE 2 Strain OD (660) Valine (g/l) FERM BP-1763 8.4 11.9 FERMBP-1763_gltAD5V 7.8 12.6

German patent application 102006032634.2, filed Jul. 13, 2006, and U.S.provisional application 60/830,331, filed Jul. 13, 2006, areincorporated herein by reference.

Numerous modification and variations on the present invention arepossible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described herein.

1. An isolated polynucleotide, encoding a polypeptide comprising theamino acid sequence of SEQ ID NO: 2, wherein L-aspartic acid at position5 of the amino acid sequence is replaced by L-valine, and wherein thepolypeptide possesses a citrate synthase activity.
 2. The isolatedpolynucleotide of claim 1, wherein the isolated polynucleotide comprisesthe nucleotide sequence of SEQ ID NO:
 5. 3. A vector comprising thepolynucleotide as claimed in claim
 1. 4. A bacterium that has beentransformed with the vector as claimed in claim 3, wherein the bacteriumis Corynebacterium glutamicum.
 5. A bacterium of Corynebacteriumglutamicum, which comprises the polynucleotide as claimed in claim
 1. 6.A recombinant bacterium of Corynebacterium glutamicum comprising apolynucleotide that codes for a polypeptide possessing a citratesynthase activity, wherein the polypeptide comprises the amino acidsequence of SEQ ID NO: 2 in which L-aspartic acid at position 5 issubstituted with L-valine.
 7. The recombinant bacterium as claimed inclaim 6, wherein the polynucleotide comprises the nucleotide sequence ofSEQ ID NO: 5.